The tissue factor pathway inhibitor (TFPI) is a circulating plasma protein which can inhibit Factor Xa (F Xa) activity. The TFPI-Xa complex can then bind to the Factor VIIa-tissue factor (VIIa-TF) complex and block the activation of Factor X (F X) and Factor IX (F IX). VIIa- TF initiates the series of reactions referred to as the extrinsic coagulation cascade. Hemophilia A (Hem A) and Hemophilia B (Hem B) plasmas are deficient in Factor VIII (F VIII) and F IX, respectively. Even though these components are commonly thought to be part of the intrinsic coagulation cascade, the clotting time of hemophilic plasma becomes prolonged more rapidly than normal plasma with increasing dilution of tissue factor. In our experiments, rabbit brain thromboplastin (Tp) was used as a source of tissue factor. Because TFPI blocks both F Xa and the VIIa-TF activation of F X in the extrinsic pathway and because neutralizing antibodies to TFPI could increase the activation of F X in plasma, we hypothesized that neutralization of TFPI activity might correct the "dilute thromboplastin" prothrombin time (dil Tp) in normal and hemophilic plasma. To approach this question, the immunoglobulin fraction (Ig) of rabbit antibodies to TFPI (HEPG2- derived) were isolated and shown to block TFPI activity in normal human serum. They also reacted with crude TFPI material in ELISA and Western blot analyses. Dilute Tp times of Hem A, Hem B and normal plasmas were tested after the addition of anti-TFPI Ig, normal rabbit Ig or buffer. Samples were assayed with various dilutions of rabbit brain Tp. Hem A, Hem B and normal plasma dil Tp times were all reduced by anti-TFPI Ig in a dose-dependent manner. The neutralization of TFPI allowed a greater production of F Xa activity which resulted in reduction of clotting time not only in normal plasma but in Hem A and Hem B plasmas as well. Moreover, at high Ig concentrations, Hem A plasma dil Tp time was reduced to times equivalent to normal plasma. These results emphasize the importance of F X as central to the coagulation process and suggest the importance of F VIII and F IX for proper function of the extrinsic coagulation pathway.